Various types of tests related to patient diagnosis and therapy can be performed by analysis of a biological sample. Biological samples containing the patient's microorganisms are taken from a patient's infections, bodily fluids or abscesses. Microorganisms from these samples are typically placed in test panels or arrays, combined with various reagents, incubated, and analyzed to aid in treatment of the patient. Biochemical analyzers have been developed to meet the needs of health care facilities and other institutions to facilitate analysis of patient samples and to improve the accuracy and reliability of assay results when compared to analysis using manual operations.
An important family of microbiological analyzers function as a diagnostic tool for determining both the identity of an infecting microorganism and of an antibiotic effective in inhibition of growth of the microorganism. In performing these in vitro tests, identification and antibiotic susceptibility patterns of microorganisms isolated from biological samples are ascertained. Such analyzers place a small sample to be tested into a plurality of small sample test wells in panels or arrays that typically contain different enzyme substrates or antimicrobics in single or serial dilutions. Identification (ID) of microorganisms and determination of Minimum Inhibitory Concentrations (MIC) of an antibiotic effective against the microorganism are determined by color changes or the degree of cloudiness (turbidity) in the sample test wells created in the arrays. By examining the signal patterns generated, both MIC and ID determination and subsequent analysis are performed by computer controlled microbiological analyzers to provide advantages in reproducibility, reduction in processing time, avoidance of transcription errors and standardization for all tests run in the laboratory.
In ID testing of a microorganism, a standardized dilution of the microorganism sample, known as an inoculum, is first prepared in order to provide a cellular suspension having a concentration within a predetermined range. This inoculum is placed in an analytical test array or panel having a number of wells. The test wells contain predetermined identification media consisting of enzyme substrates or antibiotics, which, depending on the species of microorganism present, will exhibit color changes or increases in turbidity after incubation. For instance, bacterial genera may be identified on the basis of pH changes, its ability to utilize different carbon compounds, or growth in the presence of antimicrobial agents in a test well. Some tests require addition of reagents to detect products of microorganism metabolism while others are self-indicating. In conventional chromogenic and colorimetric panels, the inoculum is incubated for a period of time before analysis is completed. By examining the reaction of the inoculum and reagents after incubation and comparing that reaction with that of known species, the types of microorganisms can be identified. Importantly, a large number of different substrates or other reagents must be available in ID testing of an unknown microorganism because the microorganism will be more or less sensitive to different substrates and reagents. This may be achieved by providing a variety of ID test panels, each pre-loaded with substrates and reagents that are selected to produce a known pattern of measurable reaction signals for various microorganisms.
The use of microbiological test panels and the techniques employed in antibiotic susceptibility testing, AST, of microorganisms, in order to determine their MIC, is well known. AST tests are tests using wells filled with inoculum and a growth broth, called herein an inoculum-broth solution, and increasing concentrations of a number of different antibiotics as used in different AST tests to determine which antimicrobial agents are most effective against a particular microorganism. The different antimicrobial agents are typically diluted in Mueller-Hinton broth with calcium and magnesium in colorimetric panels. The antimicrobials are diluted to concentrations that include those of clinical interest. AST testing requires that the test trays be incubated at a controlled temperature for a period of time so that an observable change in the number of cells has a chance to occur. Each well of the test tray is then examined for changes in turbidity. The analyzer compares each test well reading with a threshold value. The threshold value is a fixed number corresponding to a certain percentage of relative absorbency that corresponds to clinically significant growth. These changes are interpreted using a variety of methods to identify the minimum inhibitory concentrations of various antibiotics for different microorganisms.
Analyzers that carry out multi-step biochemical analytical procedures in an automated or semi-automated fashion are well known. For example, microbiological analytical systems currently carry out automated MIC procedures using both photometric and fluorometric detection methods. The MicroScan Division of Dade Behring Inc. sells a device of this type under the trade designation WalkAway® analyzer. Armes et al. U.S. Pat. No. 4,676,951, Hanaway U.S. Pat. Nos. 4,643,879 and 4,681,741, and Masterson et al. U.S. Pat. No. 5,645,800 describe certain features of the WalkAway® analyzer. Prior commercial embodiments of the Walk-Away system analyze panels carrying microbiologic samples. Automated features of more recent microbiological testing machines are well known in the art, having been described in the following patents from which it may be seen that functions such as automated handling and transport of test devices like panels or rotors throughout an analyzer are well known. Those skilled in the art have a variety of well-known techniques and choices for the routine tasks of test device transport, optical testing, computer control, etc., as described in a number of U.S. Patents, for instance, the biochemical analyzers and ID and MIC techniques described in the following U.S. Pat. Nos. 3,928,140; 3,957,583; 4,101,383; 4,236,211; 4,448,534; and 4,453,220.
More recently, advances have been made in the art of microbial MIC and ID testing, including use of advanced light sources, and use of improved methods to enhance the accuracy of the ID and MIC determinations.
U.S. Pat. No. 5,580,784 discloses the use of chemical sensors to determine whether a particular test well is evidencing bacterial growth by directing radiation sources having closely spaced wavelengths into the well. Emissions from the chemical sensor due to the two spectrally spaced radiation sources are monitored, and a ratio of their differences and sums is calculated in order to minimize station-to-station variation between the radiation sources or detectors, and lot-to-lot variations in the sensor materials.
U.S. Pat. No. 5,593,854 discloses a method of analyzing data from a fluorescent chemical sensor by calculating a ratio based on the AC and DC components of the emission from the sensor. This ratio, or the emission modulation, changes if bacterial growth is ongoing in the test well. By focusing the desired ratio into a high resolution area, and adjusting the frequency until the system reaches that ratio, one ensures that all readings are performed at a high resolution area of the sensor. The adjusted frequency is utilized to provide an indication of whether the particular vial is experiencing bacterial growth.
U.S. Pat. No. 5,629,169 estimates drug effectiveness from a drug diffusion sample including a plate having a medium containing a test organism and a plurality of antibiotic disks positioned on the plate in a medium. An inhibition zone surrounds each of the antibiotic disks after incubation. The drug diffusion sample is illuminated, and an image of the drug diffusion sample is acquired with a video camera. The image is analyzed by determining the locations of the antibiotic disks, determining the average brightness and the brightness variance of the image in a region surrounding each of the antibiotic disks, and estimating the radius of the inhibition zone surrounding each of the antibiotic disks from the average brightness and the brightness variance. The radius of the inhibition zone is indicative of drug effectiveness.
U.S. Pat. No. 5,965,090 provides an automatic sample testing machine for testing samples stored in test cards. The machine has a test sample positioning system for moving a tray containing a plurality of test sample cards and fluid receptacles among various stations in the machine. The machine has a diluting station for adding a predetermined quantity of diluent to the receptacles. A test card transport station transports the test cards from an incubation station to an optical reading station, where transmittance and fluorescence optical testing is conducted.
U.S. Pat. No. 6,086,824 discloses an automatic sample testing machine for testing samples stored in test cards. The test sample cards are placed in a tray and a transport station transports the tray from the incubation station to an optical reading station, where the cards are removed from the tray and optical measurements (e.g., transmittance and/or fluorescence optical testing) are conducted on test wells within the card. The machine has a sample loading station where test samples are placed in fluid communication with test cards in the trays.
U.S. Pat. No. 6,096,272 discloses a diagnostic microbiological testing system and method for microorganism identification (ID) and antimicrobial susceptibility determinations (AST). The system includes multiple-well test panels capable of performing ID and AST testing on the same test panel. Each test panel is inoculated with reagents, broth-suspended organisms, and placed into the instrument system. The instrument system includes a rotating carousel for incubation and indexing, multiple light sources each emitting different wavelength light, colorimetric and fluorometric detection, barcode test panel tracking and a control processor for making determinations based on measured test data.
U.S. Pat. No. 6,372,485 provides for both microorganism identification (ID) and AST determinations. The system includes multiple-well test panels capable of performing ID and AST testing on the same test panel. Each test panel is inoculated with reagents, broth-suspended organisms, and placed into the instrument system. The instrument system includes a rotating carousel for incubation and indexing, multiple light sources each emitting different wavelength light, precision colorimetric and fluorometric detection, barcode test panel tracking and a control processor for making determinations based on measured test data. One light source includes a plurality of LEDs arranged in a linear array. Each of the LEDs junction currents is controllable to produce a predetermined illumination profile.
From this discussion of the art state in automated microbiological analyzers, it may be seen that current microbiological analyzers frequently employ complex optical or similar techniques in order to determine density patterns of samples corresponding to ID test wells and compare those patterns to predetermined ID patterns in order to be capable of accurately performing ID testing on an unknown microorganism. However, known state-of-art analyzers are generally employing measurement techniques in which signals are assigned a positive or negative value depending on whether or not a microorganism has or has not either produced a biochemical reaction that changes a test solution's color or has or has not grown in the presence of certain antimicrobial agents. Such a “has or has not” approach to ID is susceptible to errors because of the uncertainty of establishing precise cut-off range limits between positive or negative values resulting in an unwanted degree of uncertainty or inaccuracy in ID testing.